Hydrogen peroxide assay by using enhanced chemiluminescence of ...

ANALYTICA CHIMICA ACTA

ELSEVIER

Analytica Chimica Acta 327 (1996) 161-165

Hydrogen peroxide assay by using enhanced chemiluminescence of the luminol-H202--horseradish peroxidase system: Comparative studies A. Navas Diaz, F. Garcia Sanchez*, Department

of Analytical

J.A. Gonziilez Garcia

Chemistry, Faculty of Sciences, University of Malaga,

29071 Malaga,

Spain

Received 20 July 1995; accepted 23 January 1996

Abstract

p-Iodophenol, p-coumaric acid, phenol and aniline are enhancers of the luminol-H202-horseradish peroxidase chemiluminescence. These enhanced and unenhanced chemiluminescences were used to assay low concentrations of hydrogen peroxide. The curves of cherniluminescence intensity maxima against hydrogen peroxide concentration were fitted to third order polynomial regressions, which permit a very good approach to the experimental results. Double the sensitivity (RSD 5 4.3%) and a slightly lower detection limit (e.g. 0.08 pM with p-coumaric acid of 0.18 pM without enhancer) were obtained with enhanced chemiluminescence than with unenhanced chemiluminescence. Keywords:

Enhanced chemiluminescence;Luminol; Hydrogen peroxide; Horseradishperoxidase

1. Introduction Numerous methods have been proposed for the detection and determination of low concentration of hydrogen peroxide. The most sensitive are the chemiluminescence-based methods, by using peroxyoxalates, acridinium phenyl carboxylates, luminol, etc. The chemiluminescence of luminol catalyzed by different catalysts or co-oxidants has often been used in hydrogen peroxide assays. The relation between the concentration of hydrogen peroxide and the chemiluminescence (CL) is related to the catalyst or co-oxidant used [I]. With hexacyanoferrate(III) the CL is linearity proportional to the hydrogen

* Correspondingauthor. Fax: +34 52 131884. ooO3-2670/96/$15.000 1996 Elsevier Science B.V.All rights reserved PII SOOO3-2670(96)00077-3

peroxide concentration between lo-’ and 10e4M; with copper CL is not linearly proportional to hydrogen peroxide; with peroxidase the CL is proportional to the square of the hydrogen peroxide concentration. In this paper, we centered our attention in the luminol-H202-horseradish peroxidase system. This system extends the pH range of luminol chemiluminescence to lower values. The chemiluminescence of the luminol-H202horseradish peroxidase system have been used to assay peroxidase and conjugates of this enzyme [2], enhancers [3] or inhibitors [4] of this chemiluminescence, and also hydrogen peroxide and other compounds that produce hydrogen peroxide for the luminol-H202-horseradish peroxidase reaction in coupled reactions [5].

162

A. Navas Diaz et al./Analytica

On the other hand, the chemiluminescence of this system enhanced by p-iodophenol, p-coumaric acid, firefly luciferin, etc. has commonly been applied to immunoassay and determinations of peroxidase and peroxidase conjugates [6-81, enzymatic assay by using pro-enhancers or pro-anti-enhancers of this system [9,10], and in determinations of antienhancers [ 111. Also, the chemiluminescence of the system enhanced by p-iodophenol was applied to hydrogen peroxidase determination [ 12,131. This paper describes a comparative study of the determination of hydrogen peroxide by using the chemiluminescence of the luminol-H202-horseradish peroxidase system without enhancer, and enhanced by phenol, aniline, p-coumaric acid and piodophenol. In this study, different polynomial regressions were used to fit the experimental results to a calibration graph.

2. Experimental 2.1. Reagents All stock solutions were prepared in distilled and demineralized water. Luminol (5-amino-2,3-dihydro1,4_phthalazinedione) (Sigma, St. Louis, MO, USA) was prepared by dissolving 0.0913 g of luminol (97%) in a few drops of dilute NaOH solution the volume was adjusted to 50ml with u-is-HCl buffer 0.1 M (pH 8.5). Horseradish peroxidase type VI-A (Sigma) 73 U/ml was prepared in u-is-HCl buffer (pH 8.5); hydrogen peroxide (Panreac Montplet and Esteban S.A., Barcelona, Spain) was prepared from hydrogen peroxide 6% w/v by diluting with distilled and demineralized water to 0.1 M. p-Coumaric acid (Chydroxycinnamic acid) was from Sigma; p-iodophenol was purchased from Aldrich-Chemie, Steinheim, Germany; phenol was supplied by Merck, Darmstadt, Germany; and aniline was from Probus S.A. Badalona, Spain. All stock solutions of these compounds were 0.001 M in distilled and demineralized water. 2.2. Instruments The chemiluminescence out in a Pet&n-Elmer

experiments were carried LS-50 (Beaconsfield, UK)

Chimica Acta 327 (1996) 161-165

luminescence spectrometer controlled by the FLDk (FL Data Manager, from Perkin-Elmer) program with the light source switched off. The apparattt was set in the phosphorescence mode with O.OOm! delay-time and 120ms gate time. The slit-width of the emission monochromator was set at 20nn with X(em) = 425 nm and the photomultiplie voltage set to 900V. The samples were placed in ; quartz cuvette continuously stirred with a magnetic stirrer. Polynomial fits were made with the Leatherbarrow R.J. (1990) GraFit Version 2.0 (Erithacus Software) 2.3. Procedure Solutions of 0.01 M luminol and 0.1 M hydrogen peroxide (and 0.001 M enhancer in enhancec chemiluminescence) were added to a quartz cuvettc with a 0.1 M tris-HCl buffer at pH 8.4. This mixture was diluted and distilled and demineralized water to i final volume of 3 ml. The solution was continuousl! stirred with a magnetic stirrer. The chemiluminescen reaction was triggered by manual injection of 1001.1 of a 37.14 U/ml horseradish peroxidase solution through a septum. The progress of light emission was monitored and recorded. Chemiluminescencc intensity maxima were measured.

3. Results and discussion 3.1. Optimization

of parameters

The concentrations of luminol and p-iodopheno (Fig. 1) were optimized to obtain the greates chemiluminescence intensity with ; maxima 0.67 pM hydrogen peroxide concentration (Fig. 1) This luminol final concentration (66.7 @I) was usec in the chemiluminescence enhanced by phenol aniline, p-coumaric acid and for unenhanced chemi luminescence. All the measurements were made in ; tris-HCl buffer at pH 8.4. We used the same optima concentrations of phenol and aniline as in [14], il which approximately the same concentrations o luminol, horseradish peroxidase were used and pI as in our present paper, but with a 2mM hydrogen peroxide. p-Coumaric acid concentration was opti mized with the same concentration of luminol


Chimica Acta 327 (19%) 161-165

163

Table 1 Reduced &i-square value for each polynomial Iirst, second or third order

40

20

0

60

[plodophenol]

2

0

[p-Coumnric

6

Order = 1

Order = 2

Order = 3

None Phenol Aniline p-Coumaric acid p-Iodophenol

0.711 0.671 100.0 34.6 1371

0.758 0.780 20.61 13.94 184.6

0.310 0.691 4.02 4.41 28.00

3.2. Polynomial jts

8

acid]bM

160 -

2

Enhancer

horseradish peroxidase, hydrogen peroxide and pH as p-iodophenol (Fig. 1). The final concentrations in the cuvette used in the hydrogen peroxide assay were: luminol 66.67 PM, horseradish peroxidase 1.24 U/ml, tris-HCl 0.033 M at pH 8.4. Optimal concentrations for each enhancer were: p-iodophenol 16.67 PM, p-coumaric acid 0.67 pM, phenol 0.1 mM and aniline 0.333n-M. Hydrogen peroxide concentrations were measured between 0 and 0.67 PM.

Fig. 1. Chemiluminescence intensity maxima vs. concentrations of p-iodophenol and p-coumaric acid. Experimental conditions: H202 0.67 @I; luminol 66.67 FM; horseradish peroxidase 1.24 U/ ml; tris-HCl 0.033 M at pH 8.4.

0

of the

80

pi%l

4

regression

4 [HZ021 PM

6

8

Table 1 shows the reduced chi-square values for each of the polynomial fits. The third order polynomial regressions were better than polynomial regressions of the second and first order. Fig. 2 shows the calibration graphs obtained with the use of aniline

240 _

240

160 -

160 -

0

4 [HZ021 PM

6

8

0

2

4 [H202]

6

8

PM

Fig. 2. Different polynomial fits for the chemiluminescence intensity maxima of luminol enhanced by aniline vs. hydrogen peroxide concentration. First order polynomial fit (left), second order polynomial fit (center), third order polynomial fit (right). Experimental conditions: luminol 66.67 uM; horseradish peroxidase 1.24 U/ml; tris-HCI 0.033 M at pH 8.4 and aniline 0.333 n&l.

164


by using polynomial regressions of first, second and third order. Linear fits were acceptable in all cases (correlation coefficients greater than 0.9887), but the third order polynomial fit was better for low concentrations of hydrogen peroxide. Fig. 3 shows the maximal intensities of chemiluminescence against concentrations of hydrogen peroxide (in PM) for each enhanced chemiluminescence and for the unenhanced chemiluminescence with their third order polynomial regression curves. Table 2 shows the parameters of the third order polynomial fit for each enhancer and for the emission without enhancer.

240 600

400

200

0

lzl 0

2

4

PO21

6

160

80

0 IIZI 0 2

8

4

6

8

fH2021pM

PM

Table 2 Parameters of the third order polynomial fit for each enhanced a and for unenhanced chemiluminescence. The polynomials have the form:y=dx3+cx2+bx+a Enhancer

d

C

b

a

None Phenol Aniline p-Coumruic acid p-Iodophenol

0.0763 0.0590 -0.539 -0.416 -1.346

-0.690 -0.560 7.36 5.12 20.32

3.44 4.84 8.47 16.39 10.58

0.700 0.872 3.49 -0.629 0.173

3.3. Detection

limit, sensitivity

Table 3 Detection limits, sensitivities and precisions enhanced and enhanced chemiluminescences Enhancer

Aniline

Detection (n = 3)

Phenol

140/ None Phenol Aniline p-Coumaric acid p-Iodophenol 0

2

4

6

R

Sensitivities

[H202]pM

and precision

Table 3 shows the detection limits for the determination of hydrogen peroxide by using the enhanced chemiluminescence, and the enhanced chemiluminescence by p-iodophenol, phenol, aniline and p-coumaric acid. The procedure used to determine the detection limits is shows in Fig. 4.

p-Caumaric acid

p-lodophenol

Chimica Acta 327 (1996) 161-165

[H202] 1tM

were

RSD (n = 3)

(IW

(PM)

(%)

0.18 0.19 0.13 0.08 0.12

0.19a O.Ogb 0.05b o.05c 0.08”

8.7’ 4.lb l.gb 4.3’ 2.0a

at ‘3.33 uM, b2.67 PM, ‘1.33 uM

Hz%.

I

r

CL lntensi5 17.

Sensitivity (n = 3)

calculated

limit

(RSD) of the non-

Maxima

8

5.6

4 0 El 0

2

4

6

8

I Hz021 PM

Fig. 3. Chemiluminescence intensity maxima vs. hydrogen peroxide concentration for the different enhanced chemiltinescences and for the unenhanced chemiluminescence. Experimental conditions: luminol 66.67 PM; horseradish peroxidase 1.24 U/ml; trisHClO.033 M at pH 8.4. Optimal concentrations for each enhancer were: p-iodophenol 16.67 pM; p-coumatic acid 0.67 pM; phenol 0.1 mM and aniline 0.333 mM.

0

0.04

0.08

0.12

0.16

[Hz021 PM

Fig. 4. Scheme for measurement variance method.

of detection limit by a constant


We used a constant variance method similar to that shown in [15], but with some differences. To calculate this variance we take each experimental value (between 0 and 0.6667 uM H202) and add its standard deviation. The differences between experimental results (with standard deviation added) and calculated results by the third order polynomial regression were measured. The greatest positive difference between the experimental values (with standard deviation added) and calculated values was taken as the variance constant and added to the calculated signal of the blank. The detection limit was calculated from this signal by using the third order polynomial regression. Table 3 shows the sensitivities calculated for the unenhanced chemiluminescence and for the chemiluminescence enhance by phenol, aniline, p-coumaric acid and p-iodophenol. We calculated this sensitivity by dividing the standard deviation of the intensity maxima of a sample of hydrogen peroxide by the average slope of the calibration graph. We take as the average slope the slope of the linear fit. Precisions (RSD) are also shown in Table 3. These precisions were measured as relative standard deviations (RSD) of the calculated concentrations of the hydrogen peroxide by using the third order polynomial regression.

Acknowledgements We thank the Comisi6n Interministerial de Ciencia y Technologia (Projects PB93-1006 and BI0940548) for financial support.

Chimica Acta 327 (1996) 161-165

16.5

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